The long term objective of this research is to understand the projection of the vestibular efferent neurons to the vestibular periphery in the context of the fundamental molecular sensory organization of the receptor and the vestibular primary afferents. The present proposal addresses three questions; 1) What are the ultrastructural relationships between the efferent neurotransmitter acetylcholine, represented by choline acetyltransferase (CHAT), the efferent neuromodulator calcitonin gene- related peptide (CGRP): and the nicotinic (nAChR) and muscarinic (mAChR) acetylcholine receptors in the vestibular type II hair cells and primary afferent dendrites and chalices of the chinchilla and human? 2) What are the genes subserving the efferent/afferent interaction in the chinchilla and human vestibular periphery?, and 3) What are the anatomic distributions of these expressed genes in the chinchilla vestibular end- organs and primary afferent ganglia? Specific Aim 1 combines pre- and post-embedding ultrastructural localization of ChAT, CGRP, nAChR, and the mAChR subtypes. Double labeling experiments will demonstrate the relationships between these cholinergic receptors, ChAT and CGRP. Specific Aim 2 will use reverse transcription polymerase chain reaction (RT-PCR) to amplify and clone portions of the genes subserving the efferent/afferent interaction in the chinchilla and human vestibular end-organs and primary afferent ganglia after microdissection. The use of specific PCR primer sets for the alpha2,3,4,5 and beta2,3,4 nAChR subunits and all five mAChR subtypes (m1-m5) are proposed, and degenerate primers are proposed to amplify the G-protein second messengers associated with the mAChRs as well as eight of the adenylyl cyclase isoforms. Molecular characterization of the cloned RT-PCR products is also proposed to determine the type of G-proteins expressed and the differential distribution of adenylyl cyclase isoforms, as well as to determine the chinchilla sequence homology. In Specific Aim 3, in situ hybridization histochemistry will be used to anatomically localize, in the chinchilla vestibular periphery, the genes identified in Specific Aim 2. After a detailed molecular understanding of this efferent/afferent interaction pathways during vestibular compensation, after vestibular injury, or during specific vestibular disorders such as Meniere's. In addition, molecular characterization of these neurotransmitter and receptor distributions may lead to advances in the pharmacotherapy of patients with vestibular dysfunction.